Bull. Natl. Mus. Nat. Sci., Ser. B, 44(1), pp. 41–51, February 22, 2018 Floral Scent Profiles and Flower Visitors in Species of Asarum Series Sakawanum (Aristolochiaceae) Satoshi Kakishima and Yudai Okuyama* Department of Botany, National Museum of Nature and Science, Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan *E-mail: [email protected] (Received 18 August 2017; accepted 20 December 2017) Abstract To understand the potential link between the variation in floral scents and pollinators in Asarum, a diverse plant genus of Japan, we conducted analyses of floral volatile compositions as well as field monitoring of flower visitors in species of the genus series Sakawanum. We detected a remarkably large number of floral volatile compounds, and found they are dominated by aliphatics and terpenoids but poor in benzenoids. However, despite a relatively intensive effort, we failed to identify specific species of flower visitors likely contributing well for the cross pollination of these plants. Contradicting to the genetic evidence that these species are generally outcrossing, the visi- tation frequency of the winged insects for their flowers was likely to be low and thus it remained enigmatic how they successfully cross-pollinate in the wild population. Key words: Asarum, floral scent, Heterotropa, pollination, SPME. The Japan archipelago harbors a rich endemic theless, only a few species are examined for the flora and has been designated as one of the biodi- plant-pollinator interactions in the genus (Suga- versity hotspots (Boufford et al., 2005; Mitter- wara, 1988; Mesler and Lu, 1993). The paucity meier et al., 2011). There are 1862 species and of the information on pollination system of Asa- 847 varieties of endemic land plants in Japan, rum in Japan is probably due to several reasons. accounting a quarter of the plant species native to First, the frequency of pollinator visitation is not Japan (Kato and Ebihara, 2011). Although these high, second, the floral organs are enclosed with endemic plants are scattered across 515 plant calyx tube so that the pollinator behaviors are genera and 161 families, with their close rela- invisible after they entered inside the flower, and tives are often found in the surrounding area of lastly, many of the species are now endangered Japan, some plant lineages are especially species and not abundant enough to conduct field obser- rich in Japan. vations. All of the documented pollination sys- Among these, the genus Asarum is a plant tem of the genus so far, including that of A. genus with the third highest number of the tamaense in Japan, involve mushroom-feeding endemic species in Japan, followed by Carex dipterans as the principal pollinator (Sugawara, (Cyperaceae) and Cirsium (Asteraceae) (Suga- 1988), thereby mushroom mimic has been sug- wara, 2006; Okuyama, 2010; Kato and Ebihara, gested for their pollination strategy (Sinn et al., 2011). The species richness of the genus is repre- 2015). However, we noticed that the floral scents sented by the marked diversity of floral forms of the species in Japan are markedly variable and including color, shape, and size, suggesting that most of them are far from the mushroom-like varieties of plant-pollinator interactions have note, which is expected for the mushroom-mim- shaped the diversification of the genus. Never- icking flowers. Nevertheless, very little is known 42 Satoshi Kakishima and Yudai Okuyama about the variation of the floral scent profile of column (30 m × 0.25 mm; film thickness, 250 the genus, with only those of 7 species in Japan µm; Restek, Bellefonte, PA, USA). Helium was and Taiwan are documented so far (Azuma et al., used as the carrier gas at a velocity of 48.1 cm s－1, 2010). and the injector temperature was 250°C. The Here, as the initial attempt to illustrate the injector was operated in the splitless mode for diversity of the pollination systems and associ- 1 min. Electron ionization mass spectra were ated floral scent profiles within the genus, we obtained at a source temperature of 200°C. The report the flower visitors and the floral scent pro- oven temperature was programmed to the follow- files of the species of the Asarum series Sakawa- ing sequence: 40°C for 5 min, followed by an num (hereafter Sakawanum species), a well char- increase of 5°C/min to 210°C and then 10°C/min acterized small subclade of the genus in Japan. to 280°C, at which the oven was held for 5 min. The rerative peak area in the total ion chromato- Materials and Methods gram (TIC) was used as a rough estimate of the relative content of each compound in the sam- Chemical analyses ples. We studied all the species and varieties of the For every volatile compound, retention indices genus Asarum series Sakawanum, i.e., Asarum were calculated with n-alkane (C6–C20) stan- costatum, A. sakawanum var. sakawanum, A. dards (Wako, Tokyo, Japan). Then identification sakawanum var. stellatum, and A. minamitania- was made by comparing the mass spectra with num, where 3, 2, 2, 3 individuals of each were those in the libraries (NIST14 and NIST14s, sampled for floral scent analyses, respectively. National Institute of Standards and Technology, For each plant individual, 1–2 flowers (0–5 days USA) at the cutoff of 90% similarity and the after opening) was collected and placed in a retention indices with those reported in the NIST 100 ml glass vial sealed with aluminum foil. Vol- Chemistry WebBook (Linstrom and Mallard, atile compounds were collected for 30 min using 2012). Where available, the mass spectra as well head space-solid phase microextraction (HS- as the retention times for the individual com- SPME) with fibers of 100 µm Divinylbenzene/ pounds were also compared with the authentic Carboxen/Polydimethylsiloxane (DVB/CAR/ standards. PDMS; Supelco, Bellfonte, PA, USA). To distin- guish volatile compounds of flowers from those Time-lapse photography of the ambient air, the volatiles from an empty To monitor flower visitors, time-lapse photog- vial was used as the control. To examine the vol- raphy using Optio WG-4 cameras (Ricoh, Tokyo, atile compounds emitted from the vegetative Japan) were conducted at a wild population of A. part, a leaf of an individual of A. costatum costatum and A. minamitanianum. For A. costa- (TBG160624) was also cut from the petiole and tum, a total of 12 individuals were monitored on was analyzed for the volatile using the same 20–21 April and 14–16 May, 2016 at Nahari site method as for the flower. All the plants used in in Kochi prefecture (N33° 25′ 26.9″, E134° 2′ this study are cultivated in Tsukuba Botanical 13.6″). For A. minamitanianum, 3 individuals Garden of the National Museum of Nature and were monitored on 24–25 March, 2014 at Hyuga Science, Japan. site in Miyazaki Prefecture (exact location of the The samples were subjected to gas chromatog- site is not shown for conservation reasons). The raphy/mass spectrometry (GC/MS) with the camera was set in front of a plant individual, equivalent settings to those reported previously keeping ＞5 cm distance from the plants. The (Okamoto et al., 2015). Specifically, we used time-intervals between the shots were set to GCMS-QP2010SE system (Shimadzu, Kyoto, 2 min, as this is almost the minimum time-inter- Japan) equipped with an Rtx-5SilMS capillary val for camera battery to be sustained overnight Floral Scent and Flower Visitor in Asarum spp. 43 (＞20 h). Any animal individual touching the Flower visitors in the wild populations of A. upper surface of the calyx was count as a single costatum and A. minamitanianum visit, and the animal on the same flower taken in Using time-lapse photography, we monitored the subsequent shot was not count. flower visitors of A. costatum and A. minamitani- anum for 19–44 h (586–1338 photographs) per plant individual (Table 2). As the result, we Results detected 8–54 flower visits per plant individual Floral volatile compounds in Sakawanum species for A. costatum while detected only 5–8 flower We detected 35–73 volatile compounds in the visits per plant individual for A. minamitania- individual headspace samples. Overall, 133 floral num. Most (＞88%) of the flower visitors were volatile compounds were found from three Asa- flightless or ground dwelling animals (Table 3). rum species and one variety in the series Judging from the contiguous shots of the photo- Sakawanum, of which 12 were aliphatics, 11 graphs, some of the pictured animals clearly were C5-branched chain compounds including entered inside the calyx tube. This suggests their hemiterpenes, 30 were monoterpenes, 71 were potential as pollinators, although it was unclear sesquiterpenes, two were diterpenes, and one was whether the pollen attached on their body (Fig. the other class of compound, while the remaining 1). six were unidentified compounds (Table 1). Among these, methyl angelate, α-pinene, cam- Discussion phene, β-pinene, limonene, trans- and cis-β- ocimene, β-caryophyllene, trans-α-bergamotene, We detected 156 floral volatile compounds, and β-selinene were common among all samples. which is a remarkably large number for only We did not detect any benzenoids from the sam- three closely-related plant species, i.e., A. costa- ples, although they constitute a major class of tum, A. minamitanianum, and A. sakawanum. volatile compounds in most of the insect-polli- This was somewhat unexpected because the vol- nated flowers. Although the volatile composition atile compounds usually detected in a set of represented by the TIC peak area ratios was vari- closely related species are usually far less. For able among the plant individuals, there was a example, Okamoto et al. (2015) found only 27 clear tendency among the species.